Clinical Evaluation of Metagenomic Next-Generation Sequencing Method for the Diagnosis of Suspected Ascitic Infection in Patients with Liver Cirrhosis in a Clinical Laboratory

ABSTRACT Metagenomic next-generation sequencing (mNGS), mostly carried out in independent clinical laboratories, has been increasingly applied in clinical pathogen diagnosis. We aimed to explore the feasibility of mNGS in clinical laboratories and analyze its potential in the diagnosis of infectious ascites. Two reference panels composed of 12 strains commonly appearing in peritonitis were constructed to evaluate the performance metrics based on in-house mNGS protocols. The mNGS clinical detection value was analyzed in 211 ascitic samples and compared with culture and composite standards. Finally, eight patients with cirrhosis were prospectively enrolled to verify the clinical value of mNGS in peritoneal infection diagnosis. The mNGS analytical performance showed that the assay had great linearity, specificity, stability, interference, and limits of detection of 33 to 828 CFU/mL. The sensitivity and specificity of mNGS for bacterial or fungal detection using culture standards were 84.2% and 82.0%, respectively. After adjustment using digital PCR and clinical judgment, the sensitivity and specificity increased to 87.2% and 90.1%, respectively. Compared with culture, mNGS detected a broad range of pathogens and more polymicrobial infections (49% versus 9%, P < 0.05). The pathogen results were obtained within 24 h using mNGS in eight prospective cases, which effectively guided antibiotics therapy. mNGS testing in clinical laboratories affiliated with a hospital has certain advantages. It has unique superiority in pathogens detection, particularly in patients with polymicrobial infections. However, considering spectrum characteristics and test cost, pertinent pathogen panels should be developed in clinical practice. IMPORTANCE This study established and evaluated a complete metagenomics next-generation sequencing assay to improve the diagnosis of suspected ascitic infection in a clinical laboratory affiliated with a hospital. The assay is superior to traditional culture testing and will aid in the early and accurate identification of pathogens, particularly in patients with polymicrobial infections. This assay is also essential for precision therapy and can reduce the incidence of drug resistance stemming from irrational use of antibiotics.

. In line 327 the authors mention that patients with advanced liver cirrhosis have a mechanism for defense against bacterial invasion. What is this mechanism because this is not universally understood? 6. In lines 338-340 the authors describe their cooperation with an independent clinical laboratory for bioinformatics analysis as a limitation. This does not sound like a limitation. If this was a limitation the authors need to clearly state what the limitation of this collaboration was.
Reviewer #2 (Public repository details (Required)): All the metagenomics data should be available Reviewer #2 (Comments for the Author): The paper from Wu et all entitled "Clinical evaluation of metagenomic next-generation sequencing method for the diagnosis of suspected ascitic infection in patients with liver cirrhosis in a clinical laboratory". This works evaluates the use of Metagenomic next-generation sequencing (mNGS) in the clinical settings. As a proof of concept, the authors performed a study on samples obtained from ascites. The results indicate that mNGS is faster, more accurate and allows for rapid treatment of infections. However, due to the cost's panels should be designed for specific pathologies.
In material and methods.
Line 131. "sequencing data were compared with the human reference genome GRCh37 (hg19) using alignment software" --> Which alignment software?
Has the digital droplet PCR (ddPCR) method been validated previously? If the method you are using is not a commercially available, what quality controls have been used in the validation? Only the bacterial dilution? Is there an internal control for sample quality and PCR quality?
How were the samples inoculated in the microbiology laboratory? It is important to know that all the culturing was appropriate and that the negative results are not because the sample processing was not appropriate. There are not details at all about the microbiology analysis.

Results section
Patient characteristics. Could the authors explain in more detail what do they mean by "incomplete data on review" (line 182)? Do they mean that no clinical data was collected?
In general: Attention to the reference, some of them appeared label as error instead a citation Font size changes in some places along the text, please revise that is consistent all along the manuscript Bacterial names should be italics. Along the manuscript there are some errors but on the tables none of them are in italics In the figure 2 some of the figures are not clear as the writing goes on top of the figures, please correct that Staff Comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. • Manuscript: A .DOC version of the revised manuscript This is a well-constructed study with a thorough analysis of how mNGS compares with conventional culture for diagnosis of SBP. Adding digital droplet PCR and clinical adjudication as a secondary comparators given the sensitivity limitations of culture was an interesting approach. Comparison of the mNGS findings for SBP and bacterascites provides valuable information about the microbes associated with each condition. Certain conclusions are not clear and the manuscript would be strengthened with clarifications of these items. Other major and minor comments are outlined below. Major: 1. In lines 180-182 the authors state WBC is increased in SBP as compared to bacterascites and no-AFI groups. Aren't SBP patients by definition supposed to have high WBC (>250). This is expected based on how patients are categorized.
2. Are there thoughts on why there was an increase in mortality with polymicrobial infections? The authors mention that Gram negative organisms are associated with the polymicrobial infections.
Are there more details regarding this? Was it not possible to grow some of the Gram negative organisms because they are fastidious or because of pretreatment with antibiotics?
3. In lines 267-268 the authors state ddPCR had the advantage of detecting low levels of bacterial infection compared to culture and mNGS. Are the authors stating mNGS was not able to detect S. pertenkovi, M. luteus, and S. pneumoniae because of lower sensitivity as compared to ddPCR? If so, it might be worth providing analytical sensitivity and specificity data comparing ddPCR and mNGS. 4. In line 268 the authors mention that the organisms identified by ddPCR alone to be low pathogenicity. S. pneumoniae was one of the organisms identified and I would not consider this a low pathogenicity organism because it is a primary cause of SBP.
5. In lines 272-273 the authors state that mNGS detected cases faster than culture. Can the authors provide data on how much faster mNGS was with turnaround time information? This could be provided in supplementary material.
6. In lines 278-279 the authors mention that timely adjustment of antibiotics were made because of mNGS results. If antibiotics were adjusted because of mNGS results, can you provide information on how they were readjusted (e.g. narrowed, broadened, discontinued)? This could be included as supplementary material.
7. In line 306 the authors state that there is no performance evaluation of parasites due to the absence of standard strains. If this is the case, this should be listed as a limitation.
8. In lines 321-323 the authors state that concentration and characteristics of bactDNA detected by molecular technology and the entire clinical condition must be taken into account in considering the clinical significance. When referring to molecular technology is this referring to ddPCR or mNGS or both? If significance is determined by droplet PCR and expert review of each case than how does mNGS play a role? 9. In lines 329-330, the authors mention that antibiotic treatment is delayed for polymicrobial infections but do not provide an explanation. Why is this the case? 10. In lines 331-333 the authors state that patients with SBP caused by Gram-negative bacteria may be associated with mixed infections and that these patients have an increase in 6-month mortality. What are possible explanations for the increase in mortality for this group?
11. In lines 344-345 the authors state "only six patients had fungal infections in our study; therefore, it is necessary to develop specific tests for peritoneal fungal infection". Are the authors stating this because it is a limitation of mNGS to identify fungal infections or because this is a characteristic of peritoneal infections? This is not clear based on how the sentence is worded.
12. Would consider adding a section in the discussion describing the impact of mNGS on the case series of 8 prospective patients with peritonitis. Minor: 1. When referring to Gram negative or Gram positive the "G" in Gram should be capitalized because it is a proper name.
2. In line 77 "lacked of accuracy analyses" could simply be stated "lacked accuracy".
3. In line 81 it would be clearer to use the wording "long turnaround time" rather than "long period". 4. In line 101 would substitute "diagnostic value" for "diagnosis value".
5. In line 327 the authors mention that patients with advanced liver cirrhosis have a mechanism for defense against bacterial invasion. What is this mechanism because this is not universally understood?
6. In lines 338-340 the authors describe their cooperation with an independent clinical laboratory for bioinformatics analysis as a limitation. This does not sound like a limitation. If this was a limitation the authors need to clearly state what the limitation of this collaboration was.

Dear Editor and Reviewers:
Thank you for your letter and the reviewer's comments concerning our manuscript " Clinical evaluation of metagenomic next-generation sequencing method for the diagnosis of suspected ascitic infection in patients with liver cirrhosis in a clinical laboratory" (ID: Spectrum02946-22). Those comments are constructive for us to revise and improve our paper.
We have studied these comments carefully and tried our best to change and improve the manuscript. Modified portions are marked in red on the paper. The leading corrections in the form and the responses to the reviewer's comments are as follows:

In lines 180-182 the authors state WBC is increased in SBP as compared to bacterascites and no-AFI groups. Aren't SBP patients by definition supposed to have high WBC (>250). This is expected based on how patients are categorized.
Response : The diagnosis of SBP is based on neutrophil count in ascitic fluid (PMN) of >250/mm3. From the demographic data, the SBP cohort showed increased white blood cell and neutrophil counts in blood and in ascites compared with those in bacterascites and no-AFI groups (P<0.001).
We have made changes on lines 192-194 and marked. Response : Thanks very much for your comments and detailed explanation.
The increased mortality of polymicrobial infections may be related to the following reasons: 1) selected the incorrect antibiotics due to polymicrobial infection leads to a delay in treatment in clinical practice; 2) cirrhosis-associated immune dysfunction, which contributes to an increased risk of infections and mortality (1); 3) previous research has shown that polymicrobial infection is "polymicrobial biofilm formation", in which polymicrobial interactions of different pathogens lead to an increased tolerance to antimicrobial agents and enhanced polymicrobial biomass (2). Our study found an increased mortality with polymicrobial infections, and similar studies have been reported in recent years (3,4,5).
In fact, Gram-negative organisms are associated with the polymicrobial infections. There are several possible reasons for this. Firstly, more recently, there has been a shift toward Gram-positive organisms, particularly in nosocomial SBP (6). Blood culture is limited by growth inhibition by prior use of antibiotics. Secondly, the possible mechanism suggests that Gram-negative bacteria are helpful for forming the bacterial biofilm matrices that might act as scaffolds that facilitate the attachment of certain bacteria (7). However, the mechanism behind this infection remains unknown and requires further research.
We have added this part of the explanation to the Discussion of line 339-350. Response : I am sorry that I didn't make myself clear. In our opinion, mNGS was able to detect S. pertenkovi, M. luteus, and S. pneumoniae, whereas ddPCR could not detect those pathogens. However, We compared the diagnostic accuracy rate between culture, mNGS, and ddPCR methods in diagnosing SBP ( Figure 1A,B). The results showed that pathogens were detected in 50.0% Response : Thank you for pointing out a meaningful question.
S. pneumoniae was one of the pathogenic organisms identified, especially in community-acquired pleural infection (7). However, the main organisms commonly appearing in peritonitis included E. coli, E. faecalis, S. aureaus, K. pneumoniae and among others, whereas S. pneumoniae is rare in peritoneal infections (8,9,10). Our study showed that in 83 patients with organisms detected by mNGS, only three patients (4%) were complicated with S.
pneumoniae infection, none of which detected by culture. Among them, one SBP patient mixed with infection of S. aureaus was characterized by fever, elevated PMN and C-reactive protein, and was treated with teicoplanin antibiotic. Two patients did not appear clinical symptoms and were not treated, and both of them were relieved of abdominal distension and discharged. Response : Thanks for your comments. Briefly, five patients were culture-negative but mNGS-positive, antibiotics were given according to mNGS results. Two patients performed incorrect antibiotic prophylaxis use and were adjusted to appropriate antibiotic therapy according to mNGS results. One patient mixed with fungal infection and were added anti-fungal drugs for further treatment.

In lines
The detailed information of 8 patients has been included in supplementary materials.

In line 306 the authors state that there is no performance evaluation of parasites due to the absence of standard strains. If this is the case, this should be listed as a limitation.
Response : Thanks for your suggestions. The performance evaluation of parasites had been listed as a limitation in line 390-393. showed that mNGS detected a broad range of pathogens, whereas ddPCR was more rapid (4h vs. 24h) and more sensitive than mNGS in diagnosing SBP, which was consistent with recent studies (11,12). In addition, the ddPCR showed a great potential to identify and exclude the common peritoneal pathogens, whereas mNGS is more appropriate in the diagnosis of rare infections and intractable diseases.
Considering the reviewer's comments, we made the complement and changes in line 362-364.

In lines 329-330, the authors mention that antibiotic treatment is delayed for polymicrobial infections but do not provide an explanation. Why is this the case?
Response : We made an explanation about this sentence. When the results of culture showed monomicrobial infections but mNGS showed polymicrobial infections, effective antibiotic coverage of pathogens could be limited merely based on culture, leading to severe clinical outcomes. We have made changes in line 339-341 of manuscript.

In lines 331-333 the authors state that patients with SBP caused by Gram-negative bacteria may be associated with mixed infections and that these patients have an increase in 6-month mortality. What are possible explanations for the increase in mortality for this group?
Response : Thanks very much for your comments.
As mentioned above, we found that patients with SBP caused by Gram-negative bacteria may be associated with mixed infections and patients with polymicrobial infection had an increase in mortality. The possible explanations for the increase in mortality are as follows: on the one hand, from polymicrobial interaction, the process of bacterial co-aggregation enhanced the pathogenesis and evolution of peritoneal infection. On the other hand, from host immune system, liver-cirrhosis immune dysfunction may predispose the host to more severe infection by translocated bacteria. In addition, incorrect antibiotic prophylaxis use contributed to a delay in treatment.

In lines 344-345 the authors state "only six patients had fungal infections in our
study; therefore, it is necessary to develop specific tests for peritoneal fungal infection".

Are the authors stating this because it is a limitation of mNGS to identify fungal infections or because this is a characteristic of peritoneal infections? This is not clear based on how the sentence is worded.
Response : I am sorry that I didn't make myself clear.
EASL clinical guideline suppose that spontaneous fungal peritonitis is a rare, less recognized and studied complication, occurring in <5% of cases, but observational data suggest a worse prognosis (13,14,15). We did not verify the fungal infection because only 6 patients with fungal infection were detected by mNGS in our study. Fungal infections are also the characteristic of peritoneal infections, so it is necessary to develop a specific tests for peritoneal fungal infection.
We made the correction and marked in manuscript.

Would consider adding a section in the discussion describing the impact of mNGS on the case series of 8 prospective patients with peritonitis.
Response : We are very grateful for your comments and suggestions.
Through the case series of 8 prospective patients with peritonitis, we found that mNGS testing not only provides a solid basis for clinical precision medicine, but also performs better antibiotic management and further improves clinical outcomes. In addition, the average length of patient stay is 12-15 days. Recent studies have also shown that rapid and accurate identification of pathogens using nucleic acid tests decreases healthcare costs as well as mortality (16,17). However, the mNGS method is still relatively expensive compared with traditional culture.
we made the complement and changes in line 384-389.

In line 327 the authors mention that patients with advanced liver cirrhosis have a mechanism for defense against bacterial invasion. What is this mechanism because this
is not universally understood?
Response : The mechanism includes two lines for defense against bacterial invasion. The gastrointestinal barrier and gut associated lymphoid tissue immune response constitute the first line of defense, and peritoneal immune function is the crucial second line of defense to prevent SBP (2,18).

When referring to Gram negative or Gram positive the "G" in Gram should be capitalized because it is a proper name.
In line 77 "lacked of accuracy analyses" could simply be stated "lacked accuracy".
In line 81 it would be clearer to use the wording "long turnaround time" rather than "long period".
In line 101 would substitute "diagnostic value" for "diagnosis value".

In lines 338-340 the authors describe their cooperation with an independent clinical laboratory for bioinformatics analysis as a limitation. This does not sound like a limitation. If this was a limitation the authors need to clearly state what the limitation of this collaboration was.
Response : Thanks very much for your comments and detailed observation.
We have made changes and marked respectively in this manuscript.

Respond to reviewers' comments
1. Line 131. "sequencing data were compared with the human reference genome GRCh37 (hg19) using alignment software" --> Which alignment software?
Response : High-quality sequencing data were compared with the human reference genome GRCh37 (hg19) using alignment softwareBowtie2 v2.4.3 (19), enabling removal of human host sequences. Our results showed that the assay had great specificity, stability, interference, linearity of 0.97-0.99 of R squared, and limits of detection of 20-45 copies/μl. This study was published in the journal Frontiers in Cellular and Infection Microbiology (20).

Has the digital droplet PCR (ddPCR) method been validated previously
In order to perform quality controls, we detected human NAKG gene of every sample for internal control, and the assay was quality controlled using negative external controls (DNase/RNase-free water) and positive external controls (known quantities of DNA from organisms) included in every batch.

How were the samples inoculated in the microbiology laboratory? It is important to
know that all the culturing was appropriate and that the negative results are not because the sample processing was not appropriate. There are not details at all about the microbiology analysis.
Response : We are grateful for your comments.
We process the samples in the microbiology laboratory as follows: Ascitic fluid obtained from each patient was injected into automatic blood culture bottles (Aerobic, Anaerobic and Myco/F Lytic bottles, 8-10 mL per bottle) to culture aerobic bacteria, anaerobic bacteria, mycobacteria and fungus in BD BACTEC FX and BD BACTECTM 9120 Automated blood culture system. When the system showed a positive growing signal, the sample was extracted from the bottle for Gram staining or Acid-fast staining to make smear microscopy, followed by subculture on Columbia blood agar and Maconkey Aga plate at 37°C with 5% CO2 for aerobic bacteria or without oxygen for anaerobic bacteria. The detailed information has been included in supplementary methods.

Could the authors explain in more detail what do they mean by "incomplete data on review" (line 182)? Do they mean that no clinical data was collected?
Response : In our study, the clinical or laboratory data in four patients were incomplete, which included one patients with sequencing failure and three patients with missing data such as blood cell count, C-reactive protein and procalcitonin level, and among others. We have made changes in manuscript of line 102-103.  Thank you for submitting the revised version of the manuscript that the previous reviewers seem to be satisfied with. Please make sure you incorporate the comments you provided in your response letter to appropriate sections of the manuscript, as deemed appropriate.

Font size changes in some places along the text, please revise that is consistent
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